Randomized placebo controlled human volunteer trial of a live oral cholera vaccine VA1.3 for safety and immune response.

Abstract

A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men aged between 16 and 50 years from Kolkata, India. A dose of 5 x 10(9)CFU (n=186) or a placebo (n=116) containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal antibody response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively. In a subgroup, anti-CT antibody rose (> or =2-folds) in 23/30 (77%) and 6/19 (32%) in vaccine and placebo recipients, respectively. These studies demonstrate that VA1.3 at a dose of 5 x 10(9) is safe and immunogenic in adults from a cholera endemic region.

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Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Randomized placebo controlled human volunteer trial of a live oral cholera
vaccine VA1.3 for safety and immune response
D. Mahalanabisa,∗, T. Ramamurthyb, G.B. Nairb, A. Ghoshc,b, S. Shaikha, B. Sena, M. Thungapathrac,
R.K. Ghoshd, G.P. Pazhanib, R.K. Nandyb, S. Janae, S.K. Bhattacharyab
aSociety for Applied Studies, CF-198, Salt Lake City, Sector-1, Kolkata 700064, India
bNational Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India
cInstitute for Microbial Technology, Chandigarh, India
dIndian Institute of Chemical Biology, 4 Raja S.C. Mallick Road, Kolkata 700010, India
eID and BG Hospital, Beliaghata, Kolkata 700010, India
a r t i c l e i n f o
Article history:
Received 5 January 2009
Received in revised form 11 May 2009
Accepted 21 May 2009
Available online xxx
Keywords:
Live oral cholera vaccine
Human volunteer trial
ctxB gene insertion
a b s t r a c t
A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was
tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men
aged between 16 and 50 years from Kolkata, India. A dose of 5×109CFU (n=186) or a placebo (n=116)
containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two
vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal anti-
body response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively.
Inasubgroup,anti-CTantibodyrose(≥2-folds)in23/30(77%)and6/19(32%)invaccineandplaceborecip-
ients, respectively. These studies demonstrate that VA1.3 at a dose of 5×109is safe and immunogenic in
adults from a cholera endemic region.
© 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Cholera is endemic in many countries, particularly in South and
South East Asia. An effective cholera vaccine has substantial public
health relevance for these countries. A single dose live oral cholera
vaccine may also help in controlling cholera outbreaks. The only
licensed live oral cholera vaccine is CVD 103-HgR that has been
marketed is largely used for travelers [1]. Recently, its production
has been discontinued. A killed oral cholera vaccine with added B-
subunit of cholera toxin (CT) has also been licensed and marketed
(DukoralTMlicensed by SBL Vaccine, Sweden). It is administered in
two doses 1–2 weeks apart. While this vaccine was mainly used for
travelers to cholera endemic regions, in recent years it has been
successfully used in refugee camps in Uganda, Sudan and Aceh
(Indonesia) to protect at risk populations. Recently, it has also been
successfully used in an endemic area in Mozambique with high
HIV prevalence [2]. A simpler version of a killed oral cholera vac-
cine without B-subunit has however been tested and implemented
in Vietnam with reported success in reducing severe disease from
cholera [3]. A reformulated version of this vaccine is undergoing
field testing in India.
∗Corresponding author. Tel.: +91 33 23370709/23588850; fax: +91 33 23370709.
E-mail addresses: sas kolkata@vsnl.net, sascf198@yahoo.com (D. Mahalanabis).
Genetically engineered live oral vaccines, incapable of elab-
orating the cholera toxin are generally derived from clinical
non-toxigenic strains of Vibrio cholerae O1 that has the potential
to cause mild to moderate levels of diarrhoea. Though the cause
behind the expression of diarrhoea by these strains are not known,
it was thought that this could be due to the presence of unknown
diarrhoeagenic factor(s) in such strains. In an attempt to circum-
vent this problem, a new oral candidate cholera vaccine has been
developed in India from a clinical non-toxigenic strain of V. cholerae
biotype El Tor serotype Inaba, which is not only devoid of CTX
prophage but also non-reactogenic as evidenced from the rabbit
illeal loop assay. This strain was selected after screening more than
1000 clinical strains of V. cholerae O1 (4). This candidate vaccine
strain contained toxR and tcpA genes that regulate CT and helps in
colonization of V. cholerae in the human gut, respectively. Through
a series of genetic manipulations (4), the cholera toxin B-subunit
encodinggene(ctxB)ofV.choleraeO1wasintroducedintothecryp-
tic hemolysin locus (hlyA) of the strain. But before doing that, ctxB
was inserted immediate by downstream of the promoter, which
is specific for the ctx operon in wild type V. cholerae strains. In
addition, a gene encoding resistance to ampicillin was introduced,
linked to the ctxB as the marker. The resulting strain, named Vac-
cine Attempt 1.3 (VA1.3), was found to be able to produce copious
amounts of CTB. In the RITARD model, this strain was found to be
non-reactogenic and provided full protection against the challenge
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2009.05.065

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doses of both V. cholerae O1, classical and El Tor biotypes [4]. The
vaccine strain VA1.3 was susceptible to tetracycline and doxycline.
The VA1.3 cholera vaccine is different from other candidate vaccine
strains such as CVD 103-HgR 638 and Peru-15, by not having the
intact hlyA.
In a randomized placebo controlled trial, we evaluated this can-
didateoralvaccinestrainin304humanvolunteersinKolkata,India,
from 1999 to 2004 for safety and immune response. Kolkata is
known to be a highly endemic zone for cholera.
2. Methods
2.1. Participants
The study was conducted in the Clinical Trials Unit jointly
administered by the Society for Applied Studies and the National
Institute of Cholera and Enteric Diseases (ICMR); it is situated at
the Infectious Diseases Hospital in Kolkata. We recruited healthy
adultmenaged16–55yearsfromFebruary1999toDecember2004.
Medical history was recorded in a pretested form and a physician
examined them for any overt or underlying illness. A routine blood
testforhemoglobinandwhitebloodcellcountwasdone.Theywent
through counseling before they were considered for participation.
We obtained written informed consent from the volunteers prior
to enrolment. Individuals with diarrhoea, vomiting, febrile illness,
history of antibiotic use during the past one month or any chronic
illness that may affect the study were excluded.
TheClinicalTrialsUnitisacompletelyindependentandseparate
facility with independent entry and exit. It has space for 8–10 beds,
a room for counseling, a room for examination. There is a separate
and dedicated toilet facility. It has an attached clinical laboratory.
This clinical facility was also subsequently used for a phase II study
of a killed oral cholera vaccine [5].
2.2. Ethical consideration
An Expert Committee constituted by the Department of
Biotechnology (DBT), Govt. of India reviewed the toxicology,
safety, reactogenicity and immunogenicity studies in animals and
approveditforfurtherstudiesinhumanvolunteerssubjecttostatu-
tory clearances. Drug Controller, Govt. of India (DCGI) approved
the human volunteer studies protocol. Indian Council of Medi-
cal Research (ICMR) Ethical Review committee approved the trial
protocol. Initially, a dose-ranging study was conducted in 16 par-
ticipants in Kolkata before starting the vaccine trial.
2.3. Preparation of live oral vaccine
The vaccine strain (VA1.3) was grown in Luria–Bertani (LB)
medium (Difco, Detroit, USA) supplemented with 50?g/ml of
ampicillin. The log phase culture was cryopreserved in 15ml
aliquots at −80◦C with 20% sterile glycerol (Merck, Germany).
Before the vaccine trial, a vial of frozen VA1.3 seed was thawed
on ice and 1.0ml of the culture was serially diluted in sterile
phosphate buffered saline (PBS) and plated in duplicate on LB
agar containing 50?g/ml of ampicillin. The plates were incubated
for 16–18h at 37◦C. Colony counts were made in plates show-
ing 30–300 colonies and the live bacterial cells in the vaccine
stock was quantified. Randomly selected colonies were also con-
firmed as V. cholerae O1 Inaba by slide agglutination test using
commercial antisera (Denka Seiken, Tokyo, Japan). On the day of
vaccination, the bacteria cells from the vaccine stock were washed
thrice in sterile PBS and the bacterial pellet was suspended in
50ml of cold sterile sodium bicarbonate buffer to give 5×109
colony forming units (CFU). We used the same buffer of the same
amount as the placebo. Both looked identical and clear solutions.
The optical density of the bacterial suspension in the buffer was
determined and adjusted to make a stock suspension having a
predicted number of CFU/ml. The vaccine dose was confirmed by
standard dilution and plate count method on mock vaccine doses,
which was made in parallel to those administrated to the volun-
teers.
2.3.1. Vaccine administration
Thevaccineformulationgrouppreparedthevaccineandblinded
the vaccine and the placebo according to the master randomization
list.Thevaccineandtheplacebowereadministratedwithinanhour
of preparation. The volunteers were admitted the night before and
thevaccineorplacebowasadministeredinthemorning.Theywere
randomized at a 3:2 ratio into vaccine and placebo groups, respec-
tively.Amasterrandomizationlistwaspreparedbeforestartingthe
studybyapersonnotdirectlyinvolvedinthestudy.Thedoseofvac-
cine was 5×109. The study participants were not allowed to eat or
drinkforanhourbeforeandaftertheintakeofthevaccine/placebo.
Before the vaccination, 150ml of sodium bicarbonate buffer was
given to the volunteers. After 5min, 50ml of VA1.3 culture suspen-
sion in sodium bicarbonate buffer and 50ml of sodium bicarbonate
buffer alone were administered for the coded vaccine and placebo,
respectively. An investigator inspected and confirmed that all the
volunteershadtakenthevaccine/placebo.Thefirst116participants
stayed in the unit for 5 days. The remaining ones stayed for 3 days.
This change was made following recommendations from the Moni-
toringCommitteeoftheDepartmentofBiotechnology,Government
of India, overseeing the project.
2.3.2. Adverse events
Adverse event of interest was diarrhoea and vomiting. All stools
were examined and graded. The stool consistency was ranked
accordingtothreegrades:grade1,firmorsoft/mushy;grade2,thick
liquid; grade 3, watery. Three or more grade 2 stool or one or more
grade 3 stool was treated as diarrhoea. The attending physician
took decision on the severity of diarrhoea and graded them as mild
with no dehydration, moderate with some dehydration and severe
with marked dehydration. Other adverse events recorded routinely
werevomiting,fever(axillarytemperature≥98◦F),abdominalpain
or discomfort, or any other adverse event that may occur dur-
ing the study period. The primary objectives of the study were
to evaluate safety and immunogenicity of the vaccine. For safety
the proportion of subjects with diarrhoeal adverse events dur-
ing the study period and for immunogenicity, the proportion of
subjects showing 4-fold or greater rise in serum vibriocidal anti-
body titre after 14 days of vaccine administration were considered.
We evaluated all adverse events during 15 days of the study
period in both vaccine and placebo recipients, and the excretion
of the vaccine strain for 5 days for first 116 volunteers and for
3 days for the remaining ones after administration of the vac-
cine.
2.3.3. Dose-ranging studies in human volunteers
Sixteen volunteers participated in a dose-ranging study. Three
volunteers each received a dose of 4.80×105, 1.29×107, 1.50×109
and 2.49×109, respectively and four volunteers received a placebo.
Volunteers were assigned according to a randomization list pre-
pared for the dose-ranging protocol prior to starting the study.
Except one volunteer who had mild vomiting (received a dose
of 2.49×109CFU), none of the volunteers had diarrhoea, vomit-
ing, nausea, fever, abdominal pain, flatulence or any other adverse
event. None of the volunteers shed the vaccine strain in the stool.
Based on these dose-ranging studies, a dose of 5×109was chosen
for safety and immune response studies in volunteers.

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2.4. Immune response
2.4.1. Vibriocidal assay
Five millilitres of venous blood was collected from the volun-
teers for vibriocidal assay prior to (0 day) and 15 days subsequent
to vaccination. Blood group was determined with blood drawn on 0
day. Vibriocidal assay was performed with the V. cholerae O1 Inaba
(VA1.3) strain using sera collected during pre- and post-vaccine
trial following the published methods [6]. Commercially prepared
guinea pig serum was used as a complement in this study (Sigma,
St. Louis, USA). The sera (100?l) were added to 100?l of PBS in the
first well to give 2-fold dilution and the subsequent dilutions were
made reciprocally up to 4800. A 4-fold or greater increase in titre
between the 0 day and 15th day sera samples was used to signify
seroconversion. Reference rabbit antiserum against O1 Inaba was
included in each assay as a control. In addition, in every batch of
the assay, serum obtained from a healthy volunteer who never had
cholera and a high titre antiserum obtained from one of the volun-
teers in this study were included as negative and positive controls,
respectively.
2.4.2. Anti-CT assay
Enzyme-linked immunosorbent assay (ELISA) was used for the
detection of response against CT antibody. Immunoglobulin G
(IgG)-specific antibody response in the paired sera were deter-
mined against purified CT using micro titration plates (Nunc,
Denmark).ELISAwasmadefollowingtheproceduresofNandyetal.
[7] with slight modification. In brief, the micro titration plates were
coated with 0.2?g of purified CT (Sigma, St. Louis, USA) in PBS. Fol-
lowing1hincubationat37◦C,wellswerewashedandblockedwith
0.5% (w/v) bovine serum albumin (fraction V, Sigma). Next, wells
were filled with 100?l of the test serum serially double diluted
in PBS containing 0.5% BSA; the initial dilution was 1:20 in all the
samples.Followingincubationandwashing,100?lofappropriately
diluted goat anti-human IgG peroxidase labeled conjugate (Sigma)
wasaddedtoeachwellandcolourwasdevelopedwiththesubstrate
solution O-phenylenedimine dihydrochloride (Sigma) and H2O2.
Results were recorded by measuring absorbance at 490nm using
an ELISA reader (BioRad, Hercules, USA) and titre was expressed as
the reciprocal of the highest dilution of antiserum that showed an
OD490value ≥0.200 in the assay. Paired sera from a subsample of
49 volunteers (30 vaccine and 19 placebo recipients) were tested
by the ELISA. The samples for anti-CT assay were chosen by a per-
son not involved with the laboratory studies. A three digit random
number was generated between 100 and 250 and using that as the
serial number of the index case, sera of 50 consecutive ID numbers
were used for choosing the samples. Sample from one participant
was inadequate.
2.5. Excretion of vaccine strain
Stools for three consecutive days collected from the partici-
pants were examined, graded and weighed. Faecal excretion of
vaccine strain was tested using conventional cultural and molec-
ular methods. Stool specimens collected from the vaccine study
participants were collected in sterile containers and transported
to the laboratory and processed within 2h of collection. For qual-
itative analysis, the stool specimens were inoculated in alkaline
peptonewater(pH8.0)andincubatedfor6–8h,followedbystreak-
ingonthiosulfatecitratebilesaltssucrose(TCBS)agar(Eiken,Tokyo,
Japan). For quantification of the vaccine strain, 1g of the stool spec-
imen was serially diluted in sterile PBS and plated on to TCBS agar
plates. Typical sucrose fermenting colonies were tested using bio-
chemical tests and were serologically confirmed using antiserum
specific for V. cholerae O1 Inaba. PCR assay was performed using
previously published method [8] with all the isolated colonies tar-
getingctxAandctxBgenesformolecularconfirmationofthevaccine
strain.
2.6. Safety measures
A physician clinically monitored the volunteers at least twice
daily. The study participants used a specially designed commode
and the faeces were passed into disposable biohazard bags. Faeces
were decontaminated by autoclaving before disposal. Doxycycline
300mg once daily for 3 days was given to all the participants on
day 7 to clear the vaccine strain.
2.7. Sample size
Assuming50%seroconversion(i.e.,vibriocidalantibodytitrerise
of ≥4-folds after 2 weeks) as a worthwhile immune response after
a single dose of live oral cholera vaccine VA1.3, a sample size of 180
in the vaccine group will give a 95% confidence limits of 43–57.5%.
With a 3:2 randomization plan we need a total of 300 volunteers to
beenrolledinthestudy.Thisgivesusalowerlimitof95%confidence
of about 43%, which is what was reported in military recruits in
Bangkok (low socio-economic group) after administration of the
licensed live oral vaccine CVD 103-HgR [9,10].
2.8. Study organization
This vaccine was developed in three research laboratories of
the Government of India and was not driven by the industry. The
Department of Biotechnology of the Ministry of Science and Tech-
nology,GovernmentofIndiawastheleadagencywhichconstituted
several committees for overseeing the progress of the work. After
completing study on 25–30 volunteers the report was reviewed
by these committees before new volunteers could be recruited.
Fig. 1. Flow chart for the recruitment of volunteers in the study.

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Table 1
Admission features of the volunteers in Kolkata (n=304).
Vaccine group (n=188) Placebo group (n=116)
Agea
Median (quartiles)
16–25 years
26–35 years
36–45 years
>45 years
22.5 (20–28.5)
128(68%)
40(21%)
18(10%)
02(1%)
22.5 (20–28)
76(66%)
28(24%)
11(9%)
01(1%)
BMI
Median (quartiles)19.18 (17.49, 20.94)18.87 (17.35, 20.67)
Hemoglobin, g/dl
Mean (SD)
Range
12.84 (0.978)
(10.01–16.01)
13.00 (0.978)
(10.56–15.01)
Education
Years in school/college
11–17 years
6–10 years
0–5 years
34
101
53
14
62
40
Blood groups
Group O
Others
56(30%)
132
36(31%)
80
aVast majority are young men in the age group of 16–25 years.
The process took time and the study extended over several
years.
2.9. Analysis
Data entry, editing and analysis were done using statistical pro-
grammes EpiInfo Version 6 (CDC, Atlanta) and Stata Version 7.0
(Stata Corporation, 4905 Lakeway Drive, College Station, TX 77845,
USA).
3. Results
3.1. Participants
Enrolment profile of the participants for the study and immune
response studies is shown in Fig. 1. A total of 186 participants in the
vaccine group and 116 in the placebo group completed the study.
ThefeaturesoftheparticipantsaregiveninTable1.Vastmajority
of them were young men in the age group of 16–25 years and came
from low socio-economic status. Nearly 30% of them belonged to
bloodgroup‘O’.BMIlevelsweregenerallylowindicatingalessthan
ideal level of nutrition. The same is true for the hemoglobin level.
3.2. Adverse events
Adverseeventswererare(Table2).Twoparticipantsafterreceiv-
ing the vaccine had mild diarrhoea and did not require any oral or
IV hydration fluids. One of them also vomited once and the other
volunteer had mild vomiting but no diarrhoea.
Table 2
Adverse events among volunteers following one dose of vaccine or placebo.
Events Vaccine (n=186)Placebo (n=116)
Diarrhoea
Vomiting
Fever
Abdominal pain
Any other adverse events
2a
2b
0
0
0
0
0
0
0
0
Total30
aMild with no dehydration, did not require oral or I.V. fluids.
bOne or two times in total in each volunteer; no treatment required.
Table 3
Serum vibriocidal antibody titres to Vibrio cholerae O1 at baseline and day 15.
GMTa
Vaccine (n=186)Placebo (n=116)
p
Baseline
Day 15
54.16
316.1
51.32
53.5
0.79
0.001
GMFbrise 5.841.04 0.001
aGMT: geometric mean reciprocal titre.
bGMF: geometric mean reciprocal titre: folds rise over baseline.
3.3. Immune response
3.3.1. Vibriocidal antibody titre
SerumvibriocidalantibodytitrestoV.choleraeO1atbaselineand
on day 15 are shown in Table 3. Geometric mean titres at baseline
were similar between the vaccine and placebo groups. The geo-
metric mean titre on day 15 in the placebo group was similar to
the baseline. In the vaccine group the mean titre was nearly 6-
folds higher on day 15 compared to the baseline. The proportion
of participants in the vaccine or placebo group with 4-fold rise and
8-fold or higher rise are shown in Table 4. The proportion of the
participants in the vaccine group who seroconverted (i.e., with 4-
fold or higher rise in titre) was 57% (95% CI: 49–64%) compared to
only 5% (95% CI: 1.4–9.8%) in the placebo group. Rate ratio for sero-
conversion between vaccine and placebo groups was 13.1 (95% CI:
5.5–31.2%). Two volunteers among the placebo recipients had an
8-fold and three had 4-fold rise in the titre. As the volunteers come
Table 4
Rise in vibriocidal antibody titre between baseline and day 15 of vaccine or placebo
dose.
Number witha
Vaccine (n=186)Placebo (n=116)
No rise
2-fold rise
4-fold rise
8-fold rise
≥16-fold rise
Number of subjects who
seroconverted (%)a
95% confidence intervalb
55(29.6%)
26(14%)
21(11.3%)
23(12.4%)
61(33%)
93(80.2%)
18 (15.5%)
3(2.6%)
2(1.7%)
0
105(56.5%) 5(4.3%)
49.0–63.7%1.4–9.8%
aNumber of subjects with ≥4-fold rise in titres from baseline on day 15.
bBinomial, exact.

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Table 5
Rise in vibriocidal antibody titre between baseline and day 15 of vaccine or placebo
dose in volunteers with blood group O.
Number witha
Vaccine (n=56)Placebo (n=36)
No rise
2-fold rise
4-fold rise
8-fold rise
≥16-fold rise
Number who seroconverted (%)a
95% confidence intervalb
19
6
5
5
22
26
8
0
1
0
32(51%)
43.2–70.3%
1(2.8%)
0.07–14.5%
aNumber (%) of subjects with ≥4-fold rise in titre from baseline to day 15.
bBinomial, exact.
from a highly endemic region for cholera, it is not unlikely for some
of them acquiring in apparent infection with cholera and have a
booster immune response. In the subgroup of participants belong-
ingtobloodgroup‘O’(Table5),theproportionwith4-foldorhigher
rise in vibriocidal antibody titre was 51% (95% CI: 43–70%) in the
vaccine group and 3% (95% CI: 0.07–15%) in the placebo group. The
rate ratio for seroconversion between vaccine and placebo group
for blood group O participants was 19.7 (95% CI: 2.8–137.5%). Rep-
resentative sera samples were sent to the International Centre for
Diarrhoeal Diseases Research, Bangladesh (ICDDR,B) and the fold
increase in the vibriocidal titres were highly reproducible (data not
shown).
3.3.2. Anti-CT antibody titre
Anti-CTantibodiesweremeasuredatbaselineand15daysaftera
singledoseofvaccineorplaceboin49volunteers(Table6).Thirtyof
themreceivedthevaccineand19receivedplacebo.Thelineartrend
for proportionate rise in anti-CT titre is significant (chi squared for
linear trend=7.3, p=0.007 with 1 degree of freedom) with higher
proportions in the vaccine group having 2-fold and 4-fold rise. The
proportion with ≥2-fold rise in anti-CT titre in the vaccine group
was 76.7% compared to 31.6% in the placebo group (RR=2.32, 95%
CI:1.22–4.84,p=0.002).Geometricmeanfoldrisewassignificantly
higher in the vaccine group (p=0.011).
3.4. Faecal excretion of the vaccine strain
The vaccine strain was not excreted in the stool in any of the
participants.
4. Discussion
In a highly endemic region of cholera, the seroconversion rate
(i.e., ≥4-fold rise in vibriocidal titre) was impressive after a single
Table 6
Riseinanti-CTantibodiesonday15afterasingleoraldoseofvaccine(inasubgroup).
Rise in anti-CT Vaccine group*(n=30) Placebo group*(n=19)
No rise
2-fold rise
4-fold rise
≥2-fold rise
6 (20%)
13 (43%)
11 (37%)
24 (80%)
13 (68.4%)
3 (15.8%)
3 (15.8%)
6 (32%)
GMT Vaccine group+(n=30)Placebo group+(n=19)
Baseline
Day 15
GMF rise
3789
7072
1.87
5308
6614
1.24, p=0.011
GMT: geometric mean reciprocal titre.
GMF rise: geometric mean reciprocal titre: folds rise over baseline.
*Chi squared for linear trend for proportionate rise in anti-CT titre=8.3, one
degree of freedom, p=0.004.
+Rate ratio for seroconversion (i.e., ≥2-fold rise)=2.27 (1.22–4.17), p=0.002.
Table 7
Vibriocidal response (≥4-fold rise) to live oral cholera vaccines in human volunteers
from literature.
Vaccine typeStudy site Dose AgeWith ≥4-fold %
rise (n)f
CVD 103-HgRa
Thai (LSEL)e
5.18×108
Placebo
4.8×108
6.37×109
1.27×1010
2×108
Placebo
5×109
Placebo
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
20% (n=95)
7% (n=104)
33% (n=39)
43% (n=40)
48% (n=40)
75% (n=40)
10% (n=30)
57% (n=186)
4% (n=116)
CVD 103-HgRb
Thai (LSEL)
Live oral Peru-15c
Bangladesh
Live orald
VA1.3
India
aRef. [9].
bRef. [10].
cRef. [13].
dPresent study.
eLSEL: low socio-economic level.
fn: number of participants.
dose of the live oral candidate vaccine VA1.3. The cholera vaccine
CVD 103-HgR was the first licensed live oral vaccine for cholera.
It has been extensively studied both in developed and developing
countryvolunteersforimmunogenicityandsafety[1,9,10].Thisvac-
cine however has been withdrawn from the market partly because
of indifferent results on protection in a large filed trial in Indonesia
[11]. In recent years a live oral cholera vaccine Peru-15 has been
extensively tested in human volunteers, both in developed coun-
tries and in places where cholera is endemic and was found to be
safe and immunogenic both in adults and in children [12–14]. We
understand that this vaccine is now awaiting field trials for protec-
tiveefficacy.Preliminarystudiesofanotherliveoralcholeravaccine
developed in Cuba, V. cholerae 638, has undergone human volun-
teerstudiesinCuba,andfoundtobeimmunogenicbutshowedmild
reactogenicity [15,16]. This vaccine, to our knowledge, has not yet
been tested in cholera endemic regions. We compared our results
with those on volunteers from endemic areas who received either
a single dose of a licensed live oral cholera vaccine (CVD 103-HgR)
or a candidate live oral vaccine Peru-15 (Table 7). The vaccine CVD
103-HgR gave a seroconversion rate from 20% to 48% among adult
Thai volunteers of low socio-economic status [9,10]. The serocon-
versionrateafterasingledoseofcandidatevaccinePeru-15was75%
(95% CI: 59–87%) in adult volunteers from a highly endemic region
(Bangladesh). Of the two licensed oral cholera vaccines, one is an
inactivated vaccine currently recommended by WHO (DukoralTM,
licensed by SBL vaccine, Sweden). It consists four batches of heat or
formalin-killed whole-cell V. cholerae O1 of both serotypes and bio-
types (classical and El Tor), with added recombinant cholera toxin
B-subunit.InitiallyitwassuccessfullyfieldtestedinBangladeshand
was also successfully used for mass vaccination in refugee camps in
Uganda,inDarfour(Sudan)andinAceh(Indonesia)toprotectatrisk
populations from potential cholera outbreaks. As stated earlier this
vaccine was also used in an endemic area in Beria (Mozambique)
and demonstrated a protective efficacy of 89% against severe diar-
rhoea with dehydration. An important observation of public health
importance is that this study area has a high HIV prevalence [2]. A
killedwhole-cellbivalentvaccine(V.choleraeO1andO139)without
B-subunit has been licensed in Vietnam in 1997 following a tech-
nology transfer from Swedish scientists. The vaccine is safe, does
not require a buffer to administer, easy to produce and is inexpen-
sive. With another technology transfer the vaccine was produced
in India that complies with WHO guidelines on killed oral cholera
vaccine. In a phase II trial in Kolkata India [5] it was found to be
immunogenic and very recently data from a phase III trial with 2
years follow-up with this vaccine have been reported (D. Sur et al.,
Abstract:The13thInternationalConferenceonEmergingInfectious

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Please cite this article in press as: Mahalanabis D, et al. Randomized placebo controlled human volunteer trial of a live oral cholera
vaccine VA1.3 for safety and immune response. Vaccine (2009), doi:10.1016/j.vaccine.2009.05.065
ARTICLE IN PRESS
G Model
JVAC-9315;No.of Pages7
6
D. Mahalanabis et al. / Vaccine xxx (2009) xxx–xxx
Diseases in the Pacific Rim, April 6–9, 2009, Kolkata, India, p. 139).
After 2 years of follow-up the overall protective efficacy was 68%.
This vaccine is being marketed in India. The killed whole-cell oral
cholera vaccines developed so far need to be given in two doses.
In the present study, the live oral cholera vaccine VA1.3 was found
to be immunogenic and largely free of adverse events in human
volunteers after a single oral dose of 5×109in an endemic area.
It is of interest to note that these volunteers coming from a low
socio-economicgroupandlivinginahighlyendemiccityforcholera
did not excrete the vaccine strain. VA1.3 has an intact tcpA gene
which is directly correlated with colonization ability of the vaccine
strain. Moreover, in animal model study, we have shown that the
vaccine strain has the ability of colonization in higher numbers. In
someoftheliveoralcholeravaccinestudiesinadultsfromendemic
region it was shown that the faecal shedding of the vaccine strains
is generally low [10].
The serum vibriocidal antibody titre as a marker of protec-
tion has been found over four decades of epidemiological studies
and vaccine efficacy trials, as a consistent and robust correlate for
protection in endemic areas, independent of age groups. Epidemi-
ological studies were conducted in the 60s and 70s by Mosley and
colleagues in connection with a parenteral vaccine trial [17–19],
in the 80s by Glass et al. [20], and in the 90s by Clemens et al.
in connection with a field trial of an oral killed whole-cell vaccine
[21]. Based on the strong association, a strong biologic gradient, the
temporal relationship and the immunological plausibility, there is
a general consensus that high vibriocidal antibody is a good marker
of protection. Experimental studies using live oral cholera vaccine
in volunteers also demonstrated that, the degree of stimulation of
serum vibriocidal antibody following ingestion of live oral cholera
vaccine is the best correlate of the antibacterial immunity in the
intestine[22,23].Whilethiscorrelationishigh,vibriocidalantibody
titre is considered an incomplete and surrogate marker for other
immune responses that are more directly related to protection
[24]. A better definition of the specific mechanisms of immunity
to cholera is yet to be determined.
One additional finding of considerable interest is the ability of
this vaccine to induce good anti-CT antibody response in these vol-
unteers.Wespeculatethatthisaddedimmunologicalcharacteristic
may further enhance the protection in field studies. It is to be noted
that ctxB gene was incorporated in the construct of VA1.3 and is
a good producer of cholera toxin-B [4]. Classical and El Tor CTs
are immunologically distinct and this difference has an influence
on the epidemiology of cholera that led the classical strains to be
completelyreplacedbytheElTorstrainswiththeadventofthesev-
enth pandemic in 1961. In recent years, El Tor strains that produce
classical cholera toxin have replaced the prototype El Tor seventh
pandemic strains of V. cholerae in Bangladesh [25], India [26] and
other countries in Asia and Africa [27]. In Kolkata, typical El Tor CT
producers are replaced by the El Tor variant strains that produce
classical CT since 1994–1995 [26]. Use of effective cholera vaccine
is now encouraged by the WHO during large cholera outbreaks.
Considering these events, the development of VA1.3 as a vaccine
strain is suited to the events that have occurred in the epidemiol-
ogyofcholerasince,thisoralvaccinerecombinantstrainhasctxBof
the classical biotype inserted into the hemolysin locus and there-
fore would be ideally suited against the current El Tor strains of V.
cholerae which produce classical cholera toxin. We should however
note that in population studies anti-CT antibody titre is not well
correlated with protection from disease.
It has been suggested that an attenuated cholera vaccine strain
could acquire ctxA gene from a wild type V. cholerae in the intes-
tine or in the environment. We contend that even if such a transfer
were to occur the adverse consequences if any would be minimal;
for such a transfer to occur there must already be a large num-
ber of toxigenic V. cholerae present in the intestine representing
simultaneous co-infection or, toxigenic V. cholerae present in the
environment to serve as a source of ctxA. The impact of one more
toxigenic strain of V. cholerae in the environment where a single
cholera patient can excrete up to 10l of watery stool containing
107–108toxigenic V. cholerae per ml per day would be insignifi-
cant. To reduce this remote possibility further, RecA gene has been
deleted in the vaccine strain Peru-15. However CVD-103 which is
not devoid of RecA gene, has been tested in a variety of settings on
a large number of people and has an excellent safety record. It has
amply demonstrated that the consequences of the presence of the
RecA gene neither compromised its safety nor led to any adverse
consequences arising from re-acquirement of ctxA.
Like all microbes V. cholerae can indeed evolve and acquire new
attributestheemergenceofV.choleraeO139beingacaseinpoint.It
is perhaps worthwhile to note that the rare theoretical possibilities
with minimal practical consequence such as the reacquisition of
the ctxA by an attenuated cholera vaccine strain are not significant
compared to the normal evolutionary process acting on a wild type
pathogen.
Multiple studies in cholera endemic regions have shown that
individuals with blood group O are more susceptible to severe
choleraduetoclassicalandElTorV.choleraeO1aswellasV.cholerae
O139[28–32].However,twostudieshaveshownthatthereisnodif-
ference in the risk of infection (asymptomatic or mild illness) with
V. cholerae among persons with blood group O antigens from those
with other blood groups [32,33]. Vibriocidal antibody response to
the live oral cholera vaccine VA1.3 in blood group O individuals
was comparable to the overall response. Its relevance to protection
from severe disease due to cholera can only be evaluated in a field
intervention trial.
Live oral cholera vaccine VA1.3 was found to be very safe. Unto-
ward events after immunization were virtually absent. In human
volunteers from a highly endemic region the vaccine strain was not
excreted in the stool after oral immunization. The rise in vibriocidal
titre,thesurrogatemarkerofprotection,involunteersfromahighly
endemic area is comparable to the other live oral cholera vaccines
tried in endemic regions. Good anti-CT antibody response is a rele-
vant feature for a candidate vaccine for cholera. We speculate that
insertion of ctxB of the classical biotype in the hemolysin locus of
VA1.3 may be an added advantage. The findings of this study sug-
gest further studies of the candidate vaccine VA1.3 including a field
intervention trial for protective efficacy.
Acknowledgements
This work was supported by the Department of Biotechnol-
ogy, Ministry of Science and Technology, Government of India. We
acknowledge Jakir Hossain for assistance in data management and
analysis, the Principal of ID & BG Hospital, Beliaghata, Kolkata for
his support.
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vaccine VA1.3 for safety and immune response. Vaccine (2009), doi:10.1016/j.vaccine.2009.05.065
ARTICLE IN PRESS
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